[A] CoralLoad Concentrate contains 2 gel-tracking dyes for improved pipetting visibility during PCR setup, enabling immediate gel loading of PCR products for [B] easy visualization of DNA migration.|The HotStarTaq Plus procedure is fast and easy for maximum convenience.|PCR was performed with HotStarTaq Plus DNA Polymerase, HotStarTaq DNA Polymerase, and Taq DNA Polymerase from QIAGEN, and 3 hot-start PCR enzymes from the indicated suppliers. Parallel reactions were performed following the suppliers' recommendations, using 50 ng human genomic DNA. A 1.5 kb fragment of the human CFTR gene was amplified in 35 PCR cycles. M: markers.|Three different primer-template systems were amplified under the same conditions with either Taq DNA polymerase from Supplier R (R) or with HotStarTaq DNA Polymerase (H). System 1: A 1.1 kb fragment of a D-IgI homolog was amplified from human genomic DNA. System 2: A 296 bp fragment from the chromosomal region correlated with X-linked juvenile retinoschisis was amplified from human genomic DNA. System 3: A 214 bp fragment of the β-actin gene was amplified from cDNA synthesized from total RNA. M: markers. Note: Data are shown for HotStarTaq DNA Polymerase; identical sensitivity and specificity were obtained with HotStarTaq Plus DNA Polymerase.|A 1.1 kb fragment of the human interleukin 1 receptor (type II) gene was amplified from cDNA. Amplification reactions were prepared in triplicate using Taq DNA polymerase and buffer from Supplier L (No hot start); antibody-mediated hot start using enzyme and buffer from Supplier L (Antibody-mediated); and HotStarTaq DNA Polymerase and PCR Buffer from QIAGEN (HotStarTaq). M: markers. Note: Data are shown for HotStarTaq DNA Polymerase; identical sensitivity and specificity were obtained with HotStarTaq Plus DNA Polymerase.|Fragments from the murine p53 gene were amplified from genomic DNA in multiplex PCR. Parallel reactions were prepared using standard reaction conditions and an enzyme from Supplier R (No hot start) or using HotStarTaq Master Mix Kit from QIAGEN (HotStarTaq Master Mix). M: markers. Note: Data are shown for HotStarTaq Master Mix Kit; identical sensitivity and specificity were obtained with HotStarTaq Plus Master Mix Kit.|Single-cell PCR of a 500 bp fragment of the murine p53 gene was carried out in duplicate using HotStarTaq and HotStarTaq Plus DNA Polymerase. Both enzymes showed equally high specificity and sensitivity. M: markers. Note: Data are shown for HotStarTaq Plus DNA Polymerase; identical sensitivity and specificity were obtained with the HotStarTaq Plus Master Mix Kit.|Ammonium and potassium cations in QIAGEN PCR Buffers increase specificity of primer annealing. K+ binds to the phosphate groups (P–) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains a high ratio of specific-to-nonspecific primer-template binding over a wide temperature range.|

Each lot of HotStarTaq Plus DNA Polymerase is subjected to a comprehensive range of quality control tests, including a stringent PCR specificity and reproducibility assay in which low-copy targets are amplified. HotStarTaq Plus Master Mix Kit outperforms kits from other suppliers and ensures high specificity and superior performance in hot-start PCR (see figures "Highest specificity" and "Higher specificity with different primer–template systems", and table). The innovative PCR buffer provided with the kit ensures specificity over a wide range of PCR conditions, minimizing the need for optimization. CoralLoad Concentrate, also included with the kit, ensures greater convenience by improving pipetting visibility and allowing direct loading of PCR products onto a gel. CoralLoad Concentrate can be added to the PCR without affecting amplification sensitivity or specificity. PCR fragments amplified in the presence of CoralLoad Concentrate have been successfully tested for cloning and restriction digestion without prior purification.

HotStarTaq Plus DNA Polymerase, a modified form of QIAGEN Taq DNA Polymerase, is supplied in an inactive state that has no polymerase activity at ambient temperatures. This prevents extension of nonspecifically annealed primers and primer–dimers formed at low temperatures during PCR setup and the initial PCR cycle (see figures "Highest specificity" and "High specificity with different primer–template systems"). HotStarTaq Plus DNA Polymerase is activated by a short 5-minute incubation at 95°C, which can be easily incorporated into any existing thermal-cycler program.

QIAGEN PCR Buffer

QIAGEN PCR Buffer maintains specific amplification in every cycle of PCR by promoting a high ratio of specific-to-nonspecific primer binding during the annealing step in each PCR cycle (see figure "Increased specificity of primer annealing"). Owing to a uniquely balanced combination of KCl and (NH4)2SO4, the buffer provides stringent primer-annealing conditions over a wider range of annealing temperatures and Mg2+ concentrations than conventional PCR buffers. Optimization of PCR by varying the annealing temperature or the Mg2+ concentration is therefore often minimal or not required.